Project Summary This research project will test the hypothesis that sortilin is a key regulator of fibrocalcific responses in calcific aortic valve disease (CAVD) through promotion of myofibroblast-like collagen producing valvular interstitial cells (VIC) phenotype and induction of VIC-derived extracellular vesicle (EVs) calcification. The role of sortilin in CAVD has never been investigated. Our unbiased network-based systems biology approach found that sortilin network is highly significantly close to the p38 MAPK protein network. In addition, single cell RNA sequencing of sortilin-expressing VICs identified enrichment of major biological pathways, including cytoskeletal organization, vesicle transport and calcification, thus suggesting that sortilin participates in CAVD by inducing myofibroblast-like phenotype, fibrosis and calcification in VICs, previously unknown functions of sortilin. The present study will explore the role of sortilin in aortic valve calcification and focus on these key pathways in our mechanistic studies. Specific Aim 1 will test the hypothesis in vivo that sortilin accelerates fibrocalcific responses in the aortic valve. These experiments will be performed in a mouse model of calcific aortic stenosis using sortilin-deficient mice, a compound mutant strain recently established in PIs laboratory, and human aortic valve leaflets containing minimal calcification obtained from patients with CAVD. Under control of molecular imaging, the portions of these leaflets representing early CAVD (e.g., fibrosis, microcalcification) will be dissected and used for our analyses. In addition, we will employ innovative methods for detection of EV-derived microcalcifications and VIC phenotypes, including density dependent scanning electron microscopy (DD-SEM), high-resolution microscopy, nanoparticle tracking analysis, 3D-bioprinted hydrogel platform, proteomics, single cell analyses, and complex network analyses. Specific Aim 2 will test the hypothesis in vitro that sortilin mediates VIC fibrocalcific response by promoting VIC myofibroblast-like phenotype, collagen production, and the release and mineralization of EVs; further aggregation of EVs within newly formed collagen fibers results in the formation of microcalcifications. We propose that sortilin induces VIC fibrocalcific responses and that genetic deletion of sortilin will decrease collagen production and retard the formation of microcalcifications in human VICs and mice. To facilitate clinical translation of mouse data, we will employ human primary VICs and aortic valve specimens from patients with CAVD. These complementary studies will advance the field by examining the role of sortilin in early CAVD. In the long-term, the findings from this project will identify novel molecular determinants that drive CAVD, and define new targets for much-needed therapies for patients with this devastating disorder.